Compositions and Methods for Preventing Cardiac Arrhythmia

ABSTRACT

Disclosed herein are compositions and methods for treating or preventing cardiac arrhythmia in a subject.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a division of U.S. application Ser. No. 12/707,427,filed on Feb. 17, 2010, which claims the benefit of U.S. ProvisionalApplication No. 61/153,402, filed on Feb. 18, 2009.

BACKGROUND OF THE INVENTION

Cardiac arrhythmias present a significant health problem. Cardiacarrhythmias include but are not limited to ventricular tachycardias,supraventricular tachycardias, and atrial fibrillation. Of these, atrialfibrillation is the most common cardiac arrhythmia.

It has been estimated that over one million people in the United Statesalone suffer from atrial fibrillation. The incidence of atrialfibrillation is expected to increase over the next several decades aspopulations in the United States and Europe trend older because atrialfibrillation tends to become more common with increasing age.

Arrhythmias after cardiac surgery are a major cause of morbidity andmortality. Tolerability of arrhythmia is less in the postoperativeperiod than for similar arrhythmias in the preoperative period.Hemodynamic instability is more likely due to the possibility ofmyocardial dysfunction. Cardiopulmonary bypass, injury to the conductionsystem during surgery, metabolic and electrolyte abnormalities,especially hypokalemia and hypomagnesemia, contribute to the increasedincidence of postoperative arrhythmias. Stress of the surgery withenhanced sympathetic tone and use of inotropic support are addedfactors. Delayed arrhythmia can occur due to scar-related re-entry.

Atrial fibrillation can be treated with medication intended to maintainnormal sinus rhythm and/or decrease ventricular response rates.Specifically, many of the past attempts have been confined topharmacotherapy, radiofrequency ablation, use of implantable devices,and related approaches. While drug therapy remains a popular route forreducing some arrhythmic events, there has been recognition thatsystemic effects are often poorly tolerated. Moreover, there is beliefthat proarrhythmic tendencies exhibited by many drugs can increasemortality in many situations. It would be desirable to have moreeffective methods for treating or preventing cardiac arrhythmias.

BRIEF SUMMARY OF THE INVENTION

In accordance with the purpose of this invention, as embodied andbroadly described herein, this invention relates to compositions andmethods for treating or preventing cardiac arrhythmia in a subject.Additional advantages of the disclosed methods and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed methods and compositions.

The advantages of the disclosed methods and compositions will berealized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the invention as claimed.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed methods and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the followingdescription.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a peptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the peptide are discussed, each and every combination andpermutation of peptide and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed methods and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present methods andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinence ofthe cited documents.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds, reference to “thecompound” is a reference to one or more compounds and equivalentsthereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data are provided in a number of differentformats, and that these data represent endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units is also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to” and is not intended toexclude, for example, other additives, components, integers or steps.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

B. Methods

Disclosed herein are methods of treating or preventing a cardiacarrhythmia in a subject. The methods can comprise administering to thecardiac tissue of the subject a therapeutically effective amount of acomposition comprising a mammalian extracellular matrix (ECM).

In some aspects, the mammalian ECM is derived from a native source. Insome aspects, the mammalian ECM is produced in vitro using mammaliancells. In some aspects, the mammalian ECM is extracted directly frommammalian tissue/organs. In some aspects the composition comprisingmammalian ECM further comprises synthetic ECM.

In some aspects, the composition comprising a mammalian ECM inhibitsscar formation. In some aspects, the composition comprising a mammalianECM promotes regeneration of damaged tissue. In some aspects, thecomposition comprising a mammalian ECM inhibits inflammation.

By “treatment” is meant the medical management of a patient with theintent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

By “prevent” or “preventing” is meant reducing the frequency or severityof a disease or condition. The term does not require an absolutepreclusion of the disease or condition. Rather, this term includesdecreasing the chance for disease occurrence. Thus, disclosed aremethods of reducing the occurrence and/or severity of a cardiacarrhythmia in a subject, comprising administering to cardiac tissue ofthe subject a therapeutically effective amount of a compositioncomprising a mammalian ECM.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses, symptoms, or sequelae of a disease or disorder. Suchamelioration only requires a reduction or alteration, not necessarilyelimination, of the cause, symptom, or sequelae of a disease ordisorder.

As used herein, the term “cardiac tissue” includes the myocardium,epicardium, endocardium, and pericardium of the heart. The term as usedherein also refers to the great vessels leading to or from the heart.The term as used herein also refers to portions of the vagus nerve thatinnervate the heart.

Thus, in some aspects, the methods comprise administering a compositioncomprising a mammalian ECM to the heart of the subject. In some aspects,the methods comprise administering a composition comprising a mammalianECM to the myocardium of the subject. The myocardium can be ventricularmyocardium. The myocardium can be atrial myocardium. In some aspects,the methods comprise administering a composition comprising a mammalianECM to the epicardium of the subject. In some aspects, the methodscomprise administering a composition comprising a mammalian ECM to theendocardium of the subject. In some aspects, the methods compriseadministering a composition comprising a mammalian ECM to thepericardium of the subject.

In some aspects, the methods comprise administering a compositioncomprising a mammalian ECM to a great vessel of the subject. In someaspects, the vessel is the superior vena cava, inferior vena cava,pulmonary vein, pulmonary artery, or aorta of the subject. For example,the method can comprise administering a composition comprising amammalian ECM to the adventitia (external portion) of one or more of thegreat vessels. In some aspects, the method comprises administering acomposition comprising a mammalian ECM to the cardiac circulation. Thus,the method comprises administering a composition comprising a mammalianECM into a blood vessel or heart chamber.

Parasympathetic innervation of the heart is mediated by the vagus nerve.The right vagus innervates the sinoatrial (SA) node. Parasympathetichyperstimulation predisposes those affected to bradyarrhythmias. Theleft vagus when hyperstimulated predisposes the heart toatrioventricular (AV) blocks. Thus, in some aspects, the methodscomprise administering a composition comprising a mammalian ECM to aportion of the vagus nerve of the subject that innervates the heart.

As used herein, the term “subject” means any individual who is thetarget of administration. The subject can be a vertebrate, for example,a mammal. Thus, the subject can be a human. The term does not denote aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are intended to be covered. A patientrefers to a subject afflicted with a disease or disorder. The term“patient” includes human and veterinary subjects. As used herein, theterms “patient” and “subject” can be used interchangeably.

In some aspects, the subject of the disclosed method has been identifiedas being at risk of developing a cardiac arrhythmia. In some aspects,the subject of the disclosed method has undergone heart surgery,including, but not limited to, open-heart surgery. In some aspects, thesubject of the disclosed method has undergone multiple combined heartprocedures, including, but not limited to, open heart procedures. Insome aspects, the subject of the disclosed method has undergone heartvalve surgery. In some aspects, the subject of the disclosed method isat least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85years of age. In some aspects, the composition is administered to asubject who has had a myocardial infarction. In some aspects, thesubject of the disclosed method has emphysema or chronic obstructivepulmonary disease. In some aspects, the subject of the disclosed methodhas a history of arrhythmia.

In some aspects, the disclosed method does not comprise administering apatch comprising small intestinal submucosa (SIS) to an opening in thepericardial sac of the heart. In some aspects, the disclosed method doesnot consist of administering a patch comprising small intestinalsubmucosa (SIS) to an opening in the pericardial sac of the heart. Insome aspects, the mammalian ECM is not SIS. Thus, in some aspects, thecomposition comprising mammalian ECM does not consist of SIS. In someaspects, the composition comprising mammalian ECM is not a patch. Insome aspects, the disclosed method does not comprise administering thecomposition comprising mammalian ECM as a patch to an opening in thepericardial sac of the heart. In some aspects, the cardiac tissue of thedisclosed method is not pericardium. In some aspects, the disclosedmethod does not comprise administering the composition to thepericardium.

In other aspects, however, the disclosed method comprises administeringa patch comprising small intestinal submucosa (SIS) to an opening in thepericardial sac of the heart. The compositions used in the disclosedmethods can comprise an additional agent, such as anti-arrhythmia drugs,or non-native cells. In other aspects, the disclosed method comprisesadministering a patch comprising small intestinal submucosa (SIS) to anopening in the pericardial sac of the heart, but the method furthercomprises additional steps,

Also disclosed herein is a method of treating or preventing a cardiacarrhythmia in a subject, comprising administering to cardiac tissue ofthe subject a therapeutically effective amount of a compositioncomprising a mammalian extracellular matrix and further comprising ananti-arrhythmic drug, a lipid-lowering drug, cells, a protein, or acombination thereof.

1. Cardiac Arrhythmia

Cardiac arrhythmia (also referred to as dysrhythmia) is a term for anyof a large and heterogeneous group of conditions in which there isabnormal electrical activity in the heart. The heart beat (pulse) can betoo fast or too slow and can be regular or irregular.

Some arrhythmias are life-threatening medical emergencies that canresult in cardiac arrest and sudden death. Others cause symptoms such asan abnormal awareness of heart beat (palpitations) and can be merelyannoying. Others may not be associated with any symptoms at all butpre-dispose toward potentially life threatening stroke or embolus.

The term sinus arrhythmia refers to a normal phenomenon of mildacceleration and slowing of the heart rate that occurs with breathing inand out. It is usually quite pronounced in children, and steadilylessens with age. This can also present during meditation breathingexercises that involve deep inhaling and breath holding patterns.

Each heart beat originates as an electrical impulse from a small area oftissue in the right atrium of the heart called the sinus node orsinoatrial (SA) node. The impulse initially causes both of the atria tocontract, then activates the atrioventricular (or AV) node which isnormally the only electrical connection between the atria and theventricles or main pumping chambers. The impulse then spreads throughboth ventricles via the His Purkinje fibers causing a synchronizedcontraction of the heart muscle.

A heart rate less than 60 beats per minute is a bradycardia. This can becaused by a slowed signal from the sinus node (termed sinusbradycardia), a pause in the normal activity of the sinus node (termedsinus arrest), or by blocking of the electrical impulse on its way fromthe atria to the ventricles (termed AV block or heart block). Heartblock comes in varying degrees and severity. It can be caused byreversible poisoning of the AV node (with drugs that impair conduction)or by irreversible damage to the node.

A heart rate faster than 100 beats per minute is a tachycardia.Tachycardia can result in palpitation; however, tachycardia is notnecessarily an arrhythmia. Increased heart rate is a normal response tophysical exercise or emotional stress. This is mediated by thesympathetic nervous system's effect on the sinus node, and is calledsinus tachycardia. Other things that increase sympathetic nervous systemactivity in the heart include ingested or injected substances such ascaffeine or amphetamines, and an overactive thyroid gland(hyperthyroidism). Heart rate can be increased with sympathomimeticdrugs.

Tachycardia that is not sinus tachycardia usually results from theaddition of abnormal impulses that can begin by one of three mechanisms:automaticity, re-entry or triggered activity.

Automaticity refers to a cardiac muscle cell firing off an impulse onits own. All of the cells in the heart have the ability to initiate anaction potential; however, only some of these cells are designed toroutinely trigger heart beats. These cells are found in the conductionsystem of the heart and include the SA node, AV node, Bundle of HIS andPurkinje fibers. The SA node is a single specialized location in theatrium which has a higher automaticity (a faster pacemaker) than therest of the heart and therefore is usually responsible for setting theheart rate and initiating each heart beat. Any part of the heart thatinitiates an impulse without waiting for the SA node is called anectopic focus and is by definition a pathological phenomenon. This cancause a single premature beat now and then, or, if the ectopic focusfires more often than the SA node, it can produce a sustained abnormalrhythm. Conditions that increase automaticity include sympatheticnervous system stimulation and hypoxia. The resulting heart rhythmdepends on where the first signal begins. If it is the SA node, therhythm remains normal but rapid; if it is an ectopic focus, many typesof arrhythmia can result.

Re-entry arrhythmias occur when an electrical impulse recurrentlytravels in a tight circle within the heart, rather than moving from oneend of the heart to the other and then stopping. Every cardiac cell isable to transmit impulses in every direction but can only do so oncewithin a short period of time. Normally, the action potential impulsewill spread through the heart quickly enough that each cell will onlyrespond once. However, if conduction is abnormally slow in some areas,part of the impulse will arrive late and potentially be treated as a newimpulse. Depending on the timing, this can produce a sustained abnormalcircuit rhythm. Re-entry circuits are responsible for atrial flutter,most paroxysmal supraventricular tachycardias, and dangerous ventriculartachycardia. When an entire chamber of the heart is involved in multiplemicro-reentry circuits and therefore quivering with chaotic electricalimpulses, it is said to be in fibrillation.

Fibrillation can affect the atrium (atrial fibrillation) or theventricle (ventricular fibrillation). If left untreated, ventricularfibrillation (VF, or V-fib) can lead to death within minutes.

Triggered beats occur when problems at the level of the ion channels inindividual heart cells result in abnormal propagation of electricalactivity and can lead to sustained abnormal rhythm. Triggered beats arerelatively rare but can result from the action of anti-arrhythmic drugs.

Arrhythmia can be classified by rate (normal, tachycardia, bradycardia),or mechanism (automaticity, re-entry, fibrillation).

It is also appropriate to classify arrhythmia by site of origin. Forexample, atrial arrhythmias include premature atrial contractions(PACs), wandering atrial pacemaker, multifocal atrial tachycardia,atrial flutter, and atrial fibrillation (Afib). Junctional arrhythmiasinclude supraventricular tachycardia (SVT), AV nodal reentranttachycardia (the most common cause of paroxysmal supra-ventriculartachycardia (PSVT)), junctional rhythm, junctional tachycardia, andpremature junctional complex. Atrioventricular arrhythmias include AVreentrant tachycardia (occurs when a re-entry circuit crosses betweenthe atria and ventricles somewhere other than the AV node).

Ventricular Arrhythmias include Premature Ventricular Contractions (PVC)(sometimes called Ventricular Extra Beats (VEBs)), Acceleratedidioventricular rhythm, Monomorphic Ventricular tachycardia, Polymorphicventricular tachycardia, and Ventricular fibrillation.

Heart blocks (also known as AV blocks, the most common causes ofbradycardia) include First degree heart block (PR interval greater than200 ms in length on the surface ECG), Second degree heart block (Type 1and 2), and Third degree heart block (also known as complete heartblock).

Cardiac arrhythmias are often first detected by auscultation of theheartbeat with a stethoscope or by feeling peripheral pulses. Thesemethods cannot usually diagnose specific arrhythmias but can give ageneral indication of the heart rate and whether it is regular orirregular. Not all of the electrical impulses of the heart produceaudible or palpable beats; in many cardiac arrhythmias, the premature orabnormal beats do not produce an effective pumping action and areexperienced as “skipped” beats.

The simplest specific diagnostic test for assessment of heart rhythm isthe electrocardiogram (abbreviated ECG or EKG). A Holter monitor is anEKG recorded over a 24-hour period, to detect arrhythmias that canhappen briefly and unpredictably throughout the day.

Sudden arrhythmia death syndrome (SADS) is a term used to describesudden death due to cardiac arrest brought on by an arrhythmia. Often,the subject has no symptoms before dying suddenly. The most common causeof sudden death in the United States is coronary artery disease.Approximately 300,000 people die suddenly of this cause every year inthe United States. SADS can also be caused by, for example, manyinherited conditions and heart diseases that can affect young people.

In children, for example, viral myocarditis, long Q-T syndrome, Brugadasyndrome, Catecholaminergic polymorphic ventricular tachycardia andhypertrophic cardiomyopathy, and arrhythmogenic right ventriculardysplasia can cause SADS.

In some aspects, the cardiac arrhythmia is atrial fibrillation orventricular fibrillation.

2. Administration

In some aspects, the mammalian ECM is a patch in a form such as a sheet,plug, a laminate, a weave, a polymer matrix, a plurality of strands, orone or more strips. Thus, in some aspects, the mammalian ECM is placedinto direct contact with the cardiac tissue of a subject during heartsurgery. In some aspects, the composition comprising a mammalian ECM isadministered to an opening in the pericardial sac of the heart. In someaspects, the composition overlaps the opening in the pericardial sac.Thus, the composition comprising a mammalian ECM can be administered tothe surgical opening of the pericardium during or after heart surgery.In another aspect, the mammalian ECM can be placed into contact withcardiac structures, such as the great vessels, e.g., aorta, pulmonaryartery, pulmonary vein, superior vena cava, and inferior vena cava.

Wherein the mammalian ECM is in a solid form such as a sheet, a plug, alaminate, a weave, a polymer matrix, a plurality of strands, or one ormore strips, the composition can be attached to the cardiac tissue usingstandard means available in the art. For example, the compositioncomprising mammalian ECM can be attached to the cardiac tissue withsutures, bioadhesives such as fibrin glue, staples, and the like.

The disclosed compounds and compositions comprising a mammalian ECM canbe administered in any suitable manner. For example, the compositionscan be administered parenterally (e.g., intramuscular injection),topically or the like. Thus, in some aspects, the composition comprisinga mammalian ECM is injectable. The disclosed compositions can beinjected into the cardiac tissue using ordinary means. For example, thecomposition comprising a mammalian ECM can be delivered to the cardiactissue via a syringe or a cardiac or coronary catheter. Cardiaccatheterization (heart cath) is the insertion of a catheter into achamber or vessel of the heart. This can be done for both diagnosticand/or interventional purposes. Coronary catheterization is a subset ofthis technique, involving the catheterization of the coronary arteries.

Thus, in some aspects, the composition comprising a mammalian ECM can beinjected into the myocardium of the heart. In some aspects, thecomposition comprising a mammalian ECM can be injected into theepicardium of the heart. In some aspects, the composition comprising amammalian ECM can be injected into the endocardium of the heart. In someaspects, the composition comprising a mammalian ECM can be injected intothe pericardium of the heart. In some aspects, the compositioncomprising a mammalian ECM can be injected between layers of the heart,e.g., between the pericardium and epicardium, between the epicardium andmyocardium, and between the myocardium and endocardium.

In some aspects, the composition comprising a mammalian ECM can beadministered to the atrial or ventricular septum of the subject. Forexample, in some aspects, the composition comprising a mammalian ECM canbe administered to a ventricular septal defect. A ventricular septaldefect (VSD) is a defect in the ventricular septum, the wall dividingthe left and right ventricles of the heart. The ventricular septumconsists of an inferior muscular and superior membranous portion and isextensively innervated with conducting cardiomyocytes. The membranousportion, which is close to the atrioventricular node, is most commonlyaffected in adults and older children. Congenital VSDs are collectivelythe most common congenital heart defects.

In some aspects, the composition comprising a mammalian ECM can beadministered to an atrial septal defect (ASD). ASD is a form ofcongenital heart defect that enables blood flow between the left andright atria via the interatrial septum. The interatrial septum is thetissue that divides the right and left atria. Without this septum, or ifthere is a defect in this septum, it is possible for blood to travelfrom the left side of the heart to the right side of the heart, or viceversa. [1] Irrespective of interatrial communication bi-directions, thisresults in the mixing of arterial and venous blood. The mixing ofarterial and venous blood may or may not be hemodynamically significant,if even clinically significant. This mixture of blood may or may notresult in what is known as a “shunt”. The amount of shunting present, ifany, dictates hemodynamic significance (see Pathophysiology below). A“right-to-left-shunt” typically poses the more dangerous scenario (seePathophysiology below).

The mammalian ECM can be in an aerosol form. Thus, in some aspects, themammalian ECM can be sprayed on the cardiac tissue of the subject.

The mammalian ECM can be in a particulate form. Particulate mammalianECM can be administered by injecting an emulsified composition,spraying, layering, packing, dusting, painting, or other similar typesof application of the dry particulate, the liquid composition, or thesemi-solid compositions.

In some aspects, the composition is administered to the epicardialsurface of the heart. Thus, in some aspects, the composition isinjected, sprayed, or attached to the epicardial surface of the heart.

The exact amount of the compositions required can vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the disorder being treated. Thus, it is notpossible to specify an exact amount for every composition. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein. Thus,effective dosages and schedules for administering the compositions canbe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptom or disorder is affected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage can vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. Dosage can vary and can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products.

Following administration of a disclosed composition for treating,inhibiting, or preventing a cardiac arrhythmia, the efficacy of themethod can be assessed in various ways well known to the skilledpractitioner. For example, one of ordinary skill in the art willunderstand that a composition disclosed herein is efficacious intreating a cardiac arrhythmia in a subject using an electrocardiogram.

The compositions disclosed herein can be administered prophylacticallyto subjects who are at risk for cardiac arrhythmia. The disclosedcompositions and methods can also be used, for example, as tools toisolate and test new drug candidates for treating or preventing cardiacarrhythmia. The disclosed compositions can also be used in a variety ofways as research tools. Other uses are disclosed, apparent from thedisclosure, and/or will be understood by those in the art.

3. Combination Therapy

The herein disclosed methods can further comprise treating the subjectwith conventional antiarrhythmia therapies. For example, there are manyclasses of antiarrhythmic medications with different mechanisms ofaction and many different individual drugs within these classes. Thus,the method can further comprise administering to the subject one or moreantiarrhythmic medications.

Some arrhythmias, e.g., atrial fibrillation, cause blood clotting withinthe heart and increase risk of embolus and stroke. Anticoagulantmedications such as warfarin and heparin, and anti-platelet drugs suchas aspirin can reduce the risk of clotting. Thus, the method can furthercomprise administering to the subject an anticoagulant.

Arrhythmias can also be treated electrically, by applying a shock acrossthe heart—either externally to the chest wall, or internally to theheart via implanted electrodes or intra-operatively. Cardioversion canbe achieved either pharmacologically or via the application of a shocksynchronized to the underlying heartbeat. It is used for treatment ofsupraventricular tachycardias. In elective cardioversion, the recipientis usually sedated or lightly anesthetized for the procedure. Forexample, atrial flutter can be treated by cardioversion. Thus, themethod can further comprise treating the subject with cardioversion.

With synchronized cardioversion, a reversion shock is delivered by wayof pads or paddles of a selected amount of electric current over apredefined number of milliseconds at the optimal moment in the cardiaccycle which corresponds to the R wave of the QRS complex on the ECG.Timing the shock to the R wave prevents the delivery of the shock duringthe vulnerable period (or relative refractory period) of the cardiaccycle, which could induce ventricular fibrillation.

Defibrillation differs from cardioversion in that the shock is notsynchronized to a cardiac cycle. It is needed for the chaotic rhythm ofventricular fibrillation and is also used for pulseless ventriculartachycardia. Often, more electricity is required for defibrillation thanfor cardioversion. Because most subjects with ventricular fibrillationare unconscious, there is generally no need for sedation. Thus, themethod can further comprise treating the subject with defibrillation.

Defibrillation or cardioversion can be accomplished by an implantablecardioverter-defibrillator (ICD). Thus, the method can further compriseadministering to the subject an ICD.

Electrical treatment of arrhythmia also includes cardiac pacing.Temporary pacing can be necessary for reversible causes of very slowheartbeats, or bradycardia, (for example, from drug overdose ormyocardial infarction). A permanent pacemaker can be placed insituations where the bradycardia is not expected to recover. Thus, themethod can further comprise administering to the subject a pacemaker.

Fine probes can in some aspects be inserted through the blood vessels tomap electrical activity from within the heart. This allows abnormalareas of conduction to be located very accurately, and subsequentlydestroyed with heat, cold, electrical or laser probes.

C. Compositions

A patch of mammalian ECM has been shown to act as a mechanical scaffoldwhile the body recruits the necessary cells to remodel and repair thecardiac tissue. Disclosed herein is the surprising ability of mammalianECM to additionally treat and/or prevent cardiac arrhythmia. Thus,disclosed herein are compositions comprising mammalian ECM for use inthe disclosed method(s) for treating or preventing cardiac arrhythmia ina subject. The disclosed compositions can be natural or synthetic. Thecompositions can be de-cellularized or comprise cells such as stemcells.

The herein disclosed compositions comprising mammalian ECM can be in theform of, for example, a patch, an emulsion, an injectable solution, agel, a fluid, a paste, a powder, a strand, a strip, a spray, a vapor, anaerosol, a cream, or a coating. The composition can further comprise oneor more additional components, including, for example, a cell, peptide,polypeptide, protein or other biological moieties. Where the compositionis a patch, it can be in a form selected from a sheet, a laminate, aweave, a polymer matrix, a plurality of strands, one or more strips, ora combination thereof.

The herein disclosed compositions comprising mammalian ECM can be madeinto a particulate and fluidized as described in U.S. Pat. No. 5,275,826to Badylak, U.S. Pat. No. 6,579,538 to Spievack, and U.S. Pat. No.6,933,326 to Griffey. Fluidized or emulsified compositions (the liquidor semi-solid forms) can be present at a certain concentration, forexample at a concentration of extracellular matrix greater than about0.001 mg/ml. The concentration of these liquid or semi-solid componentsof the extracellular matrix composition can be in a range from about0.001 mg/ml to about 200 mg/ml. The concentrations can further be foundin more specific ranges such as for example the following set of ranges:about 5 mg/ml to about 150 mg/ml, about 10 mg/ml to about 125 mg/ml,about 25 mg/ml to about 100 mg/ml, about 20 mg/ml to about 75 mg/ml,about 25 mg/ml to about 60 mg/ml, about 30 mg/ml to about 50 mg/ml, andabout 35 mg/ml to about 45 mg/ml, and about 40 mg/ml. to about 42 mg/ml.This set of ranges is exemplary and not intended to be exhaustive. It iscontemplated that any value within any of these specifically listedranges is a reasonable and useful value for a concentration of a liquid,emulsion, gel, paste or other liquid or semi-solid component of thecomposition.

1. Mammalian Extracellular Matrix

Extracellular matrix materials act as a natural scaffold for repairingsoft tissues in the body. Animal studies have shown that the originalextracellular matrix material remodels and is replaced by host tissue.Mammalian ECM is a resorbable biomaterial which has been usedsuccessfully as a xenogenic tissue graft that induces constructiveremodeling of a variety of animal tissues including blood vessels,urinary bladder, dura, abdominal wall, tendons and ligaments. Examplesof mammalian ECM include small intestine submucosa (SIS), urinarybladder submucosa (UBS), stomach submucosa (SS), and liver submucosa(LS) or liver basement membrane (LBM).

The remodeling process includes rapid neovascularization and abundantaccumulation of mesenchymal and epithelial cells that support extensivedeposition of a new extracellular matrix. The noncollagenous portion offor example, the SIS extracellular matrix is composed of variousglycoproteins, such as hyaluronic acid, heparin, dermatan andchondroitin sulfate A, as well as FGF-2 and TGF-β growth factors.

After processing, mammalian ECM can retain many of the endogenousproteins which act as growth and differentiation factors. These factorsstimulate the local environment to populate the mammalian ECM with cellsthat are then able to differentiate into the original tissue that themammalian ECM is replacing.

Mammalian ECM is a scaffold matrix of polymerized “structural” proteinsthat fit into three groups: collagens, glycoproteins, and proteoglycans(which have glycosaminoglycan repeats throughout). These moleculesactually polymerize to form the scaffold or matrix of proteins thatexists in dynamic interaction with cells and closely placed functionalproteins (either on the cells, or bound to a structural protein). Thus,mammalian ECM also includes within its matrix scaffold “functional”proteins that interact with the structural proteins and with migratingor recruited cells, such as stem cells. The matrix functional proteinsalso interact with protein-expressing cells during the life andmaintenance of the matrix scaffold itself as it rebuilds and maintainsits components. Some proteins can be both a structural and functionalprotein, depending on the protein's configuration and placement in thewhole matrix.

The ECM of, for example, cardiac tissue is made up of collagen types I(predominant), III, IV, V, and VI, combined which are 92% of the dryweight of the matrix. The ECM of cardiac tissue is also made up ofglycosaminoglycans (GAGs), which include chondroitin sulfate A and B,heparan, heparin, and hyaluronic acid. Glycoproteins such as fibronectinand entactin, proteoglycans such as decorin and perlecan, and growthfactors such as transforming growth factor beta (TGF-β), fibroblastgrowth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF),are key players in the activity of a myocardium regenerating matrix.Furthermore, the precise chemical constitution of the matrix appears toplay a role in its function, including, for example, what collagen typeis prevalent in the matrix. Thus, the outcome of any tissue regenerativeprocesses can be determined by the structural and functional componentsof the matrix scaffold that form the basis of the regenerative process.

Facilitating cell adhesion functions in ECM are cell adhesion molecules(CAMs). The CAMs can either be available endogenously or added as anadditional component of the composition. CAMs are glycoproteins lodgedin the surface of the cell membrane or transmembrane connected tocytoskeletal components of the cell. Specific CAMs include cadherinsthat are calcium dependent, and more than 30 types are known. Alsoworking as CAMs are integrins which are proteins that link thecytoskeleton of the cell in which they are lodged to the extracellularmatrix or to other cells through alpha and beta transmembrane subunitson the integrin protein. Cell migration, embryogenesis, hemostasis, andwound healing are facilitated by the integrins in the matrix. Syndecansare proteoglycans that combine with ligands for initiating cell motilityand differentiation. Immunoglobulins provide any necessary immune andinflammatory responses. Selectins promote cell-cell interactions.

i. Native Sources and Preparations

In some aspects, the mammalian ECM is derived from native source. Nativeextracellular matrix scaffolds and the proteins that form them can befound in their natural environment, i.e., the extracellular matrices ofmammals. These materials can be prepared for use in mammals in tissuegraft procedures.

In some aspects, the mammalian ECM is extracted from mammaliantissue/organs. For example, in some aspects, the mammalian ECM comprisesthe basement membrane (or transitional epithelial layer), tunicapropria, tunica submucosa, tunica muscularis, tunica serosa, or acombination thereof from a mammalian tissue source. Thus, in someaspects, the mammalian ECM comprises the basement membrane (ortransitional epithelial layer) from a mammalian tissue source. In someaspects, the mammalian ECM comprises the subjacent tunica propria from amammalian tissue source. In some aspects, the mammalian ECM comprisesthe tunica submucosa from a mammalian tissue source. In some aspects,the mammalian ECM comprises the tunica muscularis from a mammaliantissue source. In some aspects, the mammalian ECM comprises the tunicaserosa from a mammalian tissue source.

For example, small intestine submucosa (SIS) is described in U.S. Pat.No. 5,275,826; urinary bladder submucosa (UBS) is described in U.S. Pat.No. 5,554,389; stomach submucosa (SS) is described in U.S. Pat. No.6,099,567; and liver submucosa (LS) or liver basement membrane (LBM) isdescribed in U.S. Pat. No. 6,379,710, each of which is incorporatedherein by reference for teachings of how to make and use these nativeextracellular matrices.

Thus, in some aspects, the mammalian ECM of the disclosed compositionsand methods is small intestine submucosa (SIS). In some aspects, themammalian ECM of the disclosed compositions and methods is urinarybladder submucosa (UBS). In some aspects, the mammalian ECM of thedisclosed compositions and methods is stomach submucosa (SS). In someaspects, the mammalian ECM of the disclosed compositions and methods isliver submucosa (LS). In some aspects, the mammalian ECM of thedisclosed compositions and methods is liver basement membrane (LBM).

In some aspects, the mammalian ECM of the disclosed compositions andmethods is from dermis. For example, AlloDerm®, produced by LifeCellCorporation, is an acellular tissue matrix which is produced from normalhuman skin using processing techniques established to remove theepidermis and cells within the dermis without significantly altering thenormal biochemistry and molecular architecture of the connective tissuematrix. The resulting product is in a freeze-dried form allowingextended shelf life and ease of shipping without degradation or loss ofthe normal tissue matrix components. AlloDerm® can retain decorin,hyaluronic acid, chondroitin sulfates, nidogen, growth factors and otherbiochemical proteins present in normal soft tissues. Additionally,AlloDerm® can contain the basement membranes of vascular channels andthe orientation of elastin and collagen fibers of the starting dermaltissue.

In some aspects, the mammalian ECM of the disclosed compositions andmethods is from fascia. In some aspects, the mammalian ECM of thedisclosed compositions and methods is from parenchymal tissue. In someaspects, the mammalian ECM of the disclosed compositions and methods isfrom pericardium. In some aspects, the mammalian ECM of the disclosedcompositions and methods is myocardial extracellular matrix. In someaspects, the mammalian ECM of the disclosed compositions and methods isfrom decellularized heart tissue, produced, for example, by coronaryartery perfusion with detergents (Ott, et al., Nat Med. 2008 February;14(2):213-21).

In some aspects, the mammalian ECM comprises a collagen scaffold derivedfrom a mammalian tissue or organ source. The collagen scaffold frommammalian source can in some aspects comprise the basement membrane ofthe mammalian tissue source.

In some aspects, the mammalian ECM is produced in vitro. For example,the mammalian ECM can be produced from culture of mammalian cells. Themammalian ECM can be produced from proteins extracted from mammaliantissue/organs. For example, in some aspects, the mammalian ECM comprisesan artificial collagen scaffold synthesized from collagen extracted froma mammalian tissue or organ source. Collagen from mammalian sources canbe retrieved from matrix-containing tissues and used to form a matrixcomposition. Extracellular matrices can be synthesized from cellcultures as in the product manufactured by Matrigel™. In addition,dermal extracellular matrix material, subcutaneous extracellular matrixmaterial, large intestine extracellular matrix material, placentalextracellular matrix material, omentum extracellular matrix material,heart extracellular matrix material, and lung extracellular matrixmaterial, can be used, derived and preserved similarly as describedherein for the SIS, SS, LBM, and UBS materials. Other organ tissuesources of basement membrane for use in accordance with the disclosedcompositions and methods include spleen, lymph nodes, salivary glands,prostate, pancreas and other secreting glands. In general, any tissue ofa mammal that has an extracellular matrix can be used for developing anextracellular matrix component.

Collagenous matrix can be selected from a variety of commerciallyavailable collagen matrices or can be prepared from a wide variety ofnatural sources of collagen. Collagenous matrix for use in accordancewith the disclosed compositions and methods can comprise highlyconserved collagens, glycoproteins, proteoglycans, andglycosaminoglycans in their natural configuration and naturalconcentration. Collagens can be from animal sources, from plant sources,or from synthetic sources, all of which are available and standard inthe art.

The proportion of scaffold material in the composition when nativescaffold is used can be large, as the natural balance of extracellularmatrix proteins in the native scaffolds usually represents greater than90% of the extracellular matrix material by dry weight. Thus, thescaffold component of the composition by weight can be generally greaterthan 50% of the total dry weight of the composition. The scaffold cancomprise an amount of the composition by weight greater than 60%,greater than 70%, greater than 80%, greater than 82%, greater than 84%,greater than 86%, greater than 88%, greater than 90%, greater than 92%,greater than 94%, greater than 96%, and greater than 98% of the totalcomposition.

Native extracellular matrices can be prepared with care that theirbioactivity for treating or preventing cardiac arrhythmia is preservedto the greatest extent possible. Key functions that can be preservedinclude control or initiation of cell adhesion, cell migration, celldifferentiation, cell proliferation, cell death (apoptosis), stimulationof angiogenesis, proteolytic activity, enzymatic activity, cellmotility, protein and cell modulation, activation of transcriptionalevents, provision for translation events, inhibition of somebioactivities, for example inhibition of coagulation, stem cellattraction, and chemotaxis. Assays for determining these activities arestandard in the art. For example, material analysis can be used toidentify the molecules present in the material composition. Also, invitro cell adhesion tests can be conducted to make sure that the fabricor composition is capable of cell adhesion.

The disclosed compositions comprising mammalian ECM can bedecellularized in order to render them non-immunogenic. In some aspects,the decellularization process is completed with some of the key proteinfunctions retained, either by replacement of proteins incidentallyextracted with the cells, or by adding exogenous cells to the matrixcomposition after cell extraction, which cells produce or carry proteinsinvolved in treating or preventing cardiac arrhythmia.

When adding proteins to the extracellular matrix composition, theproteins can be simply added with the composition, or each protein canbe covalently linked to a molecule in the matrix. Standardprotein-molecule linking procedures can be used to accomplish thecovalent attachment.

For decellularization when starting with a source tissue/organ as asource of mammalian ECM, source tissue/organ perfusion process can beused. The source tissue/organ can be perfused with a decellularizationagent, for example 0.1% peracetic acid, rendering the organ acellular.The source tissue/organ can then be cut into portions and stored (e.g.,in aqueous environment, liquid nitrogen, cold, freeze-dried, orvacuum-pressed) for later use. Any appropriate decellularizing agent canbe used in source tissue/organ perfusion process.

With regard to submucosal tissue, extractions can be carried out nearneutral pH (in a range from about pH 5.5 to about pH 7.5) in order topreserve the presence of growth factors in the matrices. Alternatively,acidic conditions (i.e., less than pH 5.5) can be used to preserve thepresence of glycosaminoglycan components, at a temperature in a rangebetween 0 and 50 degrees centigrade. In order to regulate the acidic orbasic environment for these aqueous extractions, a buffer and chaotropicagent (generally at a concentration from about 2 M to about 8 M) can beselected, such as urea (at a concentration from about 2 M to 4 M),guanidine (at a concentration from about 2 M to about 6 M, mosttypically about 4 M), sodium chloride, magnesium chloride, and non-ionicor ionic surfactants. Urea at 2 M in pH 7.4 provides extraction of FGF-2and the glycoprotein fibronectin. Using 4 M guanidine with pH 7.4 bufferyields a fraction having transforming growth factor beta. (TGF-β).

Because of the collagenous structure of basement membrane and the desireto minimize degradation of the membrane structure during celldissociation, collagen specific enzyme activity can be minimized in theenzyme solutions used in the cell-dissociation step. For example, sourcetissue/organ can be treated with a calcium chelating agent or chaotropicagent such as a mild detergent such as Triton 100. The cell dissociationstep can also be conducted using a calcium chelating agent or chaotropicagent in the absence of an enzymatic treatment of the tissue/organ. Thecell-dissociation step can be carried out by suspending source tissueslices in an agitated solution containing about 0.05 to about 2%, moretypically about 0.1 to about 1% by weight protease, optionallycontaining a chaotropic agent or a calcium chelating agent in an amounteffective to optimize release and separation of cells from the basementmembrane without substantial degradation of the membrane matrix.

After contacting the source tissue/organ with the cell-dissociationsolution for a time sufficient to release all cells from the matrix, theresulting tissue/organ basement membrane can be rinsed one or more timeswith saline and optionally stored in a frozen hydrated state or apartially dehydrated state until used as described below. Thecell-dissociation step can require several treatments with thecell-dissociation solution to release substantially all cells from thebasement membrane. The source tissue/organ can be treated with aprotease solution to remove the component cells, and the resultingextracellular matrix material is further treated to remove or inhibitany residual enzyme activity. For example, the resulting basementmembrane can be heated or treated with one or more protease inhibitors.

Basement membrane or other native extracellular matrix scaffolds can besterilized using conventional sterilization techniques including tanningwith glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the material is preferably used. For example, itis believed that strong gamma radiation can cause loss of strength inthe graft material. Example sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation, gas plasmasterilization, and high-pressure/supercritical carbon dioxide.

ii. Synthetic ECM

Also disclosed are compositions comprising synthetic ECM for use in thedisclosed methods. Synthetic ECM for use in the disclosed compositionsand methods can be formed using synthetic molecules that polymerize muchlike native collagen and which form a scaffold environment that mimicsthe native environment of mammalian ECM scaffolds. According, suchmaterials as polyethylene terephthalate fiber (Dacron®),polytetrafluoroethylene (PTFE), glutaraldehyde-cross linked pericardium,polylactate (PLA), polyglycol (PGA), hyaluronic acid, polyethyleneglycol (PEG), polyethylene, nitinol, and collagen from non-animalsources (such as plants or synthetic collagens), can be used ascomponents of a synthetic extracellular matrix scaffold. The syntheticmaterials listed are standard in the art, and forming hydrogels andmatrix-like materials with them is also standard. Their effectivenesscan be tested in vivo as disclosed earlier, by testing in mammals, alongwith components that typically constitute native extracellular matrices,particularly the growth factors and cells responsive to them.

The extracellular matrix-like materials are described generally in Rossoet al. (Journal of Cellular Physiology 199:174-180, 2004), which isincorporated by reference herein for the teachings of how to make anduse these materials. In addition, some extracellular matrix-likematerials are listed here. Particularly useful biodegradable and/orbioabsorbable polymers include polylactides, polyglycolides,polycarprolactone, polydioxane and their random and block copolymers.Examples of specific polymers include poly D,L-lactide,polylactide-co-glycolide (85:15) and polylactide-co-glycolide (75:25).The biodegradable and/or bioabsorbable polymers used in the fibrousmatrix of the disclosed compositions and methods can have a molecularweight in the range of about 1,000 to about 8,000,000 g/mole, includingabout 4,000 to about 250,000 g/mole. The biodegradable and/orbioabsorbable fiberizable material can be a biodegradable andbioabsorbable polymer. Examples of suitable polymers can be found inBezwada, Rao S. et al. (1997) Poly(p-Dioxanone) and its copolymers, inHandbook of Biodegradable Polymers, A. J. Domb, J. Kost and D. M.Wiseman, editors, Hardwood Academic Publishers, The Netherlands, pp.29-61. The biodegradable and/or bioabsorbable polymer can contain amonomer selected from the group consisting of a glycolide, lactide,dioxanone, caprolactone, trimethylene carbonate, ethylene glycol andlysine. The material can be a random copolymer, block copolymer or blendof monomers, homopolymers, copolymers, and/or heteropolymers thatcontain these monomers. The biodegradable and/or bioabsorbable polymerscan contain bioabsorbable and biodegradable linear aliphatic polyesterssuch as polyglycolide (PGA) and its random copolymerpoly(glycolide-co-lactide-) (PGA-co-PLA). The FDA has approved thesepolymers for use in surgical applications, including medical sutures. Anadvantage of these synthetic absorbable materials is their degradabilityby simple hydrolysis of the ester backbone in aqueous environments, suchas body fluids. The degradation products are ultimately metabolized tocarbon dioxide and water or can be excreted via the kidneys. Thesepolymers are very different from cellulose-based materials, which cannotbe absorbed by the body.

Other examples of suitable biocompatible polymers are polyhydroxyalkylmethacrylates including ethylmethacrylate, and hydrogels such aspolyvinylpyrrolidone, polyacrylamides, etc. Other suitable bioabsorbablematerials are biopolymers which include collagen, gelatin, alginic acid,chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acids,polylysine and copolymers of these materials. Any glycosaminoglycan(GAG) type polymer can be used. GAGs can include, e.g., heparin,chondroitin sulfate A or B, and hyaluronic acid, or their syntheticanalogues. Any combination, copolymer, polymer or blend thereof of theabove examples is contemplated for use according to the disclosedcompositions and methods. Such bioabsorbable materials can be preparedby known methods.

Nucleic acids from any source can be used as a polymeric biomaterial.Sources include naturally occurring nucleic acids as well as synthesizednucleic acids. Nucleic acids suitable for use in the disclosedcompositions and methods include naturally occurring forms of nucleicacids, such as DNA (including the A, B and Z structures), RNA (includingmRNA, tRNA, and rRNA together or separated) and cDNA, as well as anysynthetic or artificial forms of polynucleotides. The nucleic acids usedin the disclosed compositions and methods can be modified in a varietyof ways, including by cross linking, intra-chain modifications such asmethylation and capping, and by copolymerization. Additionally, otherbeneficial molecules can be attached to the nucleic acid chains. Thenucleic acids can have naturally occurring sequences or artificialsequences. The sequence of the nucleic acid can be irrelevant for manyaspects of the disclosure. However, special sequences can be used toprevent any significant effects due to the information coding propertiesof nucleic acids, to elicit particular cellular responses or to governthe physical structure of the molecule. Nucleic acids can be used in avariety of crystalline structures both in finished biomaterials andduring their production processes. Nucleic acid crystalline structurecan be influenced by salts used with the nucleic acid. For example, Na,K, Bi, and Ca salts of DNA all have different precipitation rates anddifferent crystalline structures. Additionally, pH influencescrystalline structure of nucleic acids.

The physical properties of the nucleic acids can also be influenced bythe presence of other physical characteristics. For example, inclusionof hairpin loops can result in more elastic biomaterials or can providespecific cleavage sites. The nucleic acid polymers and copolymersproduced can be used for a variety of tissue engineering applications,including to increase tissue tensile strength, improve wound healing,speed up wound healing, as templates for tissue formation, to guidetissue formation, to stimulate nerve growth, to improve vascularizationin tissues, as a biodegradable adhesive, as device or implant coating,or to improve the function of a tissue or body part. The polymers canalso more specifically be used as sutures, scaffolds and wounddressings. The type of nucleic acid polymer or copolymer used can affectthe resulting chemical and physical structure of the polymericbiomaterial.

iii. Combinations

The herein disclosed composition can comprise combinations of mammalianECM from two or more sources or in two or more distinct forms. Thus, thedisclosed compositions can comprise any combination of native and/orsynthetic mammalian ECMs disclosed herein.

Thus, for example, the composition can comprise mammalian ECMcombinations from such sources as, for example but not limited to, smallintestine submucosa, liver basement membrane, stomach submucosa, urinarybladder submucosa, placental basement membrane, pancreatic basementmembrane, large intestine submucosa, lung interstitial membrane,respiratory tract submucosa, heart extracellular matrix, dermal matrix,and in general extracellular matrix from any mammalian fetal tissue. Anyone of these tissue sources can provide extracellular matrix that canthen be manipulated into a designated form (liquid, semi-solid, or solidform), for use in a composition.

The combinations of mammalian ECM from two or more sources can be mixedsolids, mixed liquids, mixed emulsions, mixed gels, mixed pastes, ormixed solid particulates. All of these compositions are mixtures ofextracellular matrices from two or more sources, for example mixtures ofpowders or particulates from two or more extracellular matrices,mixtures of pastes from two or more extracellular matrices, mixtures ofemulsions or gels from two or more extracellular matrices and mixturesof liquids from two or more extracellular matrices.

The compositions can be made from three mammalian tissue sources, fourmammalian tissue sources, five mammalian tissue sources, six mammaliantissue sources, and conceivably up to ten or more tissue sources. Thesetissue sources can be from the same mammal (for example the same cow,the same pig, the same rodent, the same human, etc.), the same speciesof mammal (e.g. cow, pig, rodent, human), or different species ofmammals (for example liver matrix from a pig, small intestine submucosafrom a cow, and urinary bladder submucosa from a dog, all mixed togetherin the composition).

The compositions can comprise two or more liquid matrices (fromdifferent tissue sources) combined together. The composition can be twoor more emulsion matrices (from different tissue sources) combinedtogether. The composition can be two or more particulate matrices (fromdifferent tissue sources) combined together. The composition can be aliquid mixture of two or more extracellular matrices.

For example, a composition can comprise a combination of SIS in sheet,particulate, emulsion, gel or liquid form with SS, or LBM, or UBS insheet, particulate, emulsion, gel or liquid form. For example, acomposition can comprise a combination of SS in sheet, particulate,emulsion, gel or liquid form with SIS, or LBM, or UBS in sheet,particulate, emulsion, gel or liquid form. For example, a compositioncan comprise a combination of LBM in sheet, particulate, emulsion, gelor liquid form with SS, or SIS, or UBS in sheet, particulate, emulsion,gel or liquid form. For example, a composition can comprise acombination of UBS in sheet, particulate, emulsion, gel or liquid formwith SS, or SIS, or LBM in sheet, particulate, emulsion, gel or liquidform.

The disclosed compositions can comprise combinations of mammalian ECMfrom one or more sources but in two or more distinct forms. For example,a composition can comprise a gel matrix combined with a particulatematrix. In some aspects, mammalian ECM in particulate form can be dustedonto mammalian ECM in a sheet form.

In some aspects, the composition can comprise a combination of SIS, SS,or LBM, or UBS in sheet, emulsion, gel or liquid form with SIS, SS, orLBM, or UBS in particulate form. In some aspects, the composition cancomprise a combination of SIS, SS, or LBM, or UBS in particulate,emulsion, gel or liquid form with SIS, SS, or LBM, or UBS in sheet form.In some aspects, the composition can comprise a combination of SIS, SS,or LBM, or UBS in sheet, particulate, gel or liquid form with SIS, SS,or LBM, or UBS in emulsion form. In some aspects, the composition cancomprise a combination of SIS, SS, or LBM, or UBS in sheet, particulate,emulsion, or liquid form with SIS, SS, or LBM, or UBS in gel form. Insome aspects, the composition can comprise a combination of SIS, SS, orLBM, or UBS in sheet, particulate, emulsion, or gel form with SIS, SS,or LBM, or UBS in liquid form.

As disclosed herein, the composition comprising mammalian ECM can beprepared for preferred consistency. For example, mammalian ECM can beprepared as a combination of gel and particulate in a ratio optimal toprevent dissipation into the blood stream. For example, the compositioncomprising mammalian ECM can comprise about 40% ECM in gel form andabout 60% ECM in dry particulate form. Thus, disclosed herein is acomposition comprising mammalian ECM in both gel and dry particulateforms, wherein the gel form comprises about 10, 15, 20, 25, 30, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 50% of the ECM in the composition.Thus, the dry particulate form can comprise about 90, 85, 80, 75, 70,65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50% of the ECM in thecomposition.

Selection of the concentrations of the liquid or semi-solid compositions(liquids, gels, emulsions, or pastes) is important. For example, theliquid forms can be present in a range of concentrations, from verydilute at about 0.001 mg/ml to greater concentrations of up to about 200mg/ml. The concentrations can further be found in more specific rangessuch as, for example, the following set of ranges: from about 5 mg/ml toabout 150 mg/ml, from about 10 mg/ml to about 125 mg/ml, from about 25mg/ml to about 100 mg/ml, from about 20 mg/ml to about 75 mg/ml, fromabout 25 mg/ml to about 60 mg/ml, from about 30 mg/ml to about 50 mg/ml,from about 35 mg/ml to about 45 mg/ml, and from about 40 mg/ml to about42 mg/ml. This set of ranges is exemplary and not intended to beexhaustive. It is contemplated that any value within any of thesespecifically listed ranges is a reasonable and useful value for aconcentration of a liquid or semi-solid component of the composition.

The emulsion can be more concentrated than a liquid form and can retaina shape which can be useful in applying the matrix composition tocertain parts of the body, hence its characterization as a “semi-solid”.The emulsion can be concentrated enough to form shapes like a plug orother configuration suited to the site at which the matrix compositionis being applied. Thick emulsion can be painted or otherwise applied ata site as a paste, and dusted with solid particulate on top of theemulsion. The solid particulate can be reconstituted to form theemulsion, or can be applied dry as a particulate powder which can dust aregion in the subject being treated.

Dry particulate or reconstituted particulate that forms an emulsion oftwo or more mammalian ECM can be mixed together in some proportion. Forexample, 50% of SIS can be mixed with 50% of SS in a vial. This mixturecan then be fluidized by hydrating it in a suitable buffer, for examplesaline. The hydration can be accomplished to a desired concentration ofthe mammalian ECM mixture, for example in a range from about 0.001 mg/mlto about 200 mg/ml. The concentrations can further be found in morespecific ranges such as for example the following set of ranges: fromabout 5 mg/ml to about 150 mg/ml, from about 10 mg/ml to about 125mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 20 mg/ml toabout 75 mg/ml, from about 25 mg/ml to about 60 mg/ml, from about 30mg/ml to about 50 mg/ml, from about 35 mg/ml to about 45 mg/ml, and fromabout 40 mg/ml. to about 42 mg/ml. This set of ranges is exemplary andnot intended to be exhaustive. It is contemplated that any value withinany of these specifically listed ranges is a reasonable and useful valuefor a concentration of a liquid or semi-solid component of thecomposition.

The lower the concentration of extracellular matrix the more liquid thecomposition will be. The higher the concentration of extracellularmatrix the more the composition approaches a gel-like emulsion orsemi-solid consistency. The ratio of the mixtures of the two (or more)extracellular matrices in any given composition from different sources(or the same source) can be unequal. So for example, LBM can be presentat 75% and SIS can be present at 25%, i.e., a 3:1 ratio). Any suitableratio can be used: 1:1, 1:2, 1:3, 1:4, 1:5, and so on. Where 3 or moretissue sources of extracellular matrix are represented in thecomposition, the same type of balance or imbalance in the amounts of thematrices can occur. For example, for extracellular matrix from 3sources, each source can be present in equal proportions, i.e., 1:1:1(33%/33%/33%). Alternatively, a disproportionate amount of theparticulate can be from one source, e.g., 2:1:1 (50%/25%/25%). Likewise,all three sources can be present in disproportionate amounts, e.g.,50%/30%/20%.

The two or more mammalian ECMs can be fluidized (or emulsified)separately and the fluidized or emulsified compositions mixed together.As another alternative, the two or more mammalian ECMs can be fluidizedor emulsified separately and administered separately. In addition, thetwo or more mammalian ECMs can remain in particulate solid form and bemixed together in a vial for administration as a solid combinationparticulate. Rehydration of a dry particulate mammalian ECM mixture canbe accomplished just prior to use.

2. Proteins

The disclosed compositions comprising mammalian ECM can further compriseexogenous proteins, such as those normally found in mammalian ECM. Theprotein can be a collagen, a proteoglycan, a glycosaminoglycan (GAG)chain, a glycoprotein, a growth factor, a cytokine, a cell-surfaceassociated protein, a cell adhesion molecule (CAM), an angiogenic growthfactor, an endothelial ligand, a matrikine, a matrix metalloprotease, acadherin, an immunoglobulin, a fibril collagen, a non-fibrillarcollagen, a basement membrane collagen, a multiplexin, a small-leucinerich proteoglycan, decorin, biglycan, a fibromodulin, keratocan,lumican, epiphycan, a heparan sulfate proteoglycan, perlecan, agrin,testican, syndecan, glypican, serglycin, selectin, a lectican, aggrecan,versican, nuerocan, brevican, cytoplasmic domain-44 (CD-44), macrophagestimulating factor, amyloid precursor protein, heparin, chondroitinsulfate B (dermatan sulfate), chondroitin sulfate A, heparan sulfate,hyaluronic acid, fibronectin (Fn), tenascin, elastin, fibrillin,laminin, nidogen/entactin, fibulin I, fibulin II, integrin, atransmembrane molecule, platelet derived growth factor (PDGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGF-alpha),transforming growth factor beta (TGF-β), fibroblast growth factor-2(FGF-2) (also called basic fibroblast growth factor (bFGF)),thrombospondin, osteopontin, angiotensin converting enzyme (ACE), or avascular epithelial growth factor (VEGF). This list is not intended tobe exhaustive.

Thus, the herein disclosed compositions comprising a mammalian ECM cancomprise collagen I and III, elastin, laminin, CD44, hyaluronan,syndecan, bFGF, HGF, PDGF, VEGF, Fn, tenascin, heparin, heparan sulfate,chondroitin sulfate B, integrins, decorin, TGF-β, or a combinationthereof.

3. Cells

In some aspects, the herein disclosed compositions comprising mammalianECM further comprise one or more cells. In some aspects the cells arenon-native, i.e., heterologous to the mammalian ECM. In some aspects,the cells are stem cells. A non-exhaustive list of stem cells includehuman embryonic stem cell, a fetal cardiomyocyte, a myofibroblast, amesenchymal stem cell, an autotransplanted expanded cardiomyocyte, anadipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, amyoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, anembryonic stem cell, a parenchymal cell, an epithelial cell, anendothelial cell, a mesothelial cell, a fibroblast, an osteoblast, achondrocyte, an exogenous cell, an endogenous cell, a stem cell, ahematopoietic stem cell, a pluripotent stem cell, a bone marrow-derivedprogenitor cell, a progenitor cell, a myocardial cell, a skeletal cell,a fetal cell, an embryonic cell, an undifferentiated cell, amulti-potent progenitor cell, a unipotent progenitor cell, a monocyte, acardiomyocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, acapillary endothelial cell, a xenogenic cell, an allogenic cell, anadult stem cell, and a post-natal stem cell.

In some aspects, the stem cells have the potential to differentiate intocardiac tissue cells. Thus, in some aspects, the stem cells arepluripotent. In some aspects, the stem cells are angioblasts orhemangioblasts. In some aspects, the stem cells are myoblasts. Stemcells can be derived and maintained using standard methods for stem cellculture.

4. Pharmaceuticals

The herein disclosed compositions comprising mammalian ECM can furthercomprise any known or newly discovered substance that can beadministered to the heart of a subject. For example, the hereindisclosed compositions comprising mammalian ECM can further comprise anantiarrhythmic agent. Antiarrhythmic agents are a group ofpharmaceuticals that are used to suppress fast and/or irregular rhythmsof the heart (cardiac arrhythmias).

The Vaughan Williams classification, introduced in 1970, is one of themost widely used classification schemes for antiarrhythmic agents. Thisscheme classifies a drug based on the primary mechanism of itsantiarrhythmic effect. There are five main classes in the VaughanWilliams classification of antiarrhythmic agents: Class I agentsinterfere with the sodium (Na+) channel; Class II agents areanti-sympathetic nervous system agents (most agents in this class arebeta blockers); Class III agents affect potassium (K+) efflux; Class IVagents affect calcium channels and the AV node; and Class V agents workby other or unknown mechanisms.

Class Ia agents include Quinidine, Procainamide, and Disopyramide. ClassIb agents include Lidocaine, Phenytoin, and Mexiletine. Class Ic agentsinclude Flecainide, Propafenone, and Moricizine. Class II agents includePropranolol, Esmolol, Timolol, Metoprolol, and Atenolol. Class IIIagents include Amiodarone, Sotalol, Ibutilide, and Dofetilide. Class IVagents include Verapamil, and Diltiazem. Class V agents includeAdenosine and Digoxin.

Thus, the herein disclosed compositions comprising mammalian ECM canfurther comprise one or more of Quinidine, Procainamide, Disopyramide,Lidocaine, Phenytoin, Mexiletine, Flecainide, Propafenone, Moricizine,Propranolol, Esmolol, Timolol, Metoprolol, Atenolol, Amiodarone,Sotalol, Ibutilide, Dofetilide, Verapamil, Diltiazem, Adenosine andDigoxin.

The provided compositions can further comprise one or more antibiotics(e.g., Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin,Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole,Penicillins, Tetracyclines, Trimethoprim-sulfamethoxazole, andVancomycin).

The provided compositions can further comprise one or more steroids(e.g., Andranes (e.g., Testosterone), Cholestanes (e.g., Cholesterol),Cholic acids (e.g., Cholic acid), Corticosteroids (e.g., Dexamethasone),Estraenes (e.g., Estradiol), and Pregnanes (e.g., Progesterone).

The provided compositions can further comprise one or more classes ofnarcotic and non-narcotic analgesics, including, but not limited to,Morphine, Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine,Methadone, Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone,Buprenorphine, Butorphanol, Nalbuphine, and Pentazocine.

The provided compositions can further comprise one or moreanti-inflammatory agents, including, but not limited to, Alclofenac,Alclometasone Dipropionate, Algestone Acetonide, alpha Amylase,Amcinafal, Amcinafide, Amfenac Sodium, Amiprilose Hydrochloride,Anakinra, Anirolac, Anitrazafen, Apazone, Balsalazide Disodium,Bendazac, Benoxaprofen, Benzydamine Hydrochloride, Bromelains,Broperamole, Budesonide, Carprofen, Cicloprofen, Cintazone, Cliprofen,Clobetasol Propionate, Clobetasone Butyrate, Clopirac, CloticasonePropionate, Cormethasone Acetate, Cortodoxone, Decanoate, Deflazacort,Delatestryl, Depo-Testosterone, Desonide, Desoximetasone, DexamethasoneDipropionate, Diclofenac Potassium, Diclofenac Sodium, DiflorasoneDiacetate, Diflumidone Sodium, Diflunisal, Difluprednate, Diftalone,Dimethyl Sulfoxide, Drocinonide, Endrysone, Enlimomab, Enolicam Sodium,Epirizole, Etodolac, Etofenamate, Felbinac, Fenamole, Fenbufen,Fenclofenac, Fenclorac, Fendosal, Fenpipalone, Fentiazac, Flazalone,Fluazacort, Flufenamic Acid, Flumizole, Flunisolide Acetate, Flunixin,Flunixin Meglumine, Fluocortin Butyl, Fluorometholone Acetate,Fluquazone, Flurbiprofen, Fluretofen, Fluticasone Propionate,Furaprofen, Furobufen, Halcinonide, Halobetasol Propionate, HalopredoneAcetate, Ibufenac, Ibuprofen, Ibuprofen Aluminum, Ibuprofen Piconol,Ilonidap, Indomethacin, Indomethacin Sodium, Indoprofen, Indoxole,Intrazole, Isoflupredone Acetate, Isoxepac, Isoxicam, Ketoprofen,Lofemizole Hydrochloride, Lomoxicam, Loteprednol Etabonate,Meclofenamate Sodium, Meclofenamic Acid, Meclorisone Dibutyrate,Mefenamic Acid, Mesalamine, Meseclazone, Mesterolone,Methandrostenolone, Methenolone, Methenolone Acetate, MethylprednisoloneSuleptanate, Momiflumate, Nabumetone, Nandrolone, Naproxen, NaproxenSodium, Naproxol, Nimazone, Olsalazine Sodium, Orgotein, Orpanoxin,Oxandrolane, Oxaprozin, Oxyphenbutazone, Oxymetholone, ParanylineHydrochloride, Pentosan Polysulfate Sodium, Phenbutazone SodiumGlycerate, Pirfenidone, Piroxicam, Piroxicam Cinnamate, PiroxicamOlamine, Pirprofen, Prednazate, Prifelone, Prodolic Acid, Proquazone,Proxazole, Proxazole Citrate, Rimexolone, Romazarit, Salcolex,Salnacedin, Salsalate, Sanguinarium Chloride, Seclazone, Sermetacin,Stanozolol, Sudoxicam, Sulindac, Suprofen, Talmetacin, Talniflumate,Talosalate, Tebufelone, Tenidap, Tenidap Sodium, Tenoxicam, Tesicam,Tesimide, Testosterone, Testosterone Blends, Tetrydamine, Tiopinac,Tixocortol Pivalate, Tolmetin, Tolmetin Sodium, Triclonide,Triflumidate, Zidometacin, and Zomepirac Sodium.

The provided compositions can further comprise one or morelipid-lowering drugs. As used herein, the term “lipid-lowering drug”refers to a drug that can be administered to a subject to reduce theserum levels of various heart disease-associated lipids, including, butnot limited to, cholesterol, low-density lipoprotein (LDL), verylow-density lipoprotein (VLDL), and triglycerides.

For example, the lipid-lowering drugs can be statins, including, but notlimited to, Lovastatin, Simvastatin, Atorvastatin, Fluvastatin,Pravastatin, Rosuvastatin, Cervistatin, and Pitavastatin. It iscontemplated that any statin drug, now known or developed in the future,having lipid-reducing and/or anti-inflammatory properties can be used inthe compositions described herein.

The provided compositions can further comprise one or moreanti-histaminic agents, including, but not limited to, Ethanolamines(like diphenhydramine carbinoxamine), Ethylenediamine (liketripelennamine pyrilamine), Alkylamine (like chlorpheniramine,dexchlorpheniramine, brompheniramine, triprolidine), astemizole,loratadine, fexofenadine, Bropheniramine, Clemastine, Acetaminophen,Pseudoephedrine, and Triprolidine.

The provided compositions can further comprise one or moreantineoplastic drugs, including, but not limited to, Acivicin,Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin,Altretamine, Ambomycin, Ametantrone Acetate, Aminoglutethimide,Amsacrine, Anastrozole, Anthramycin, Asparaginase, Asperlin,Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide,Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, BleomycinSulfate, Brequinar Sodium, Bropirimine, Busulfan, Cactinomycin,Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, CarubicinHydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin,Cisplatin, Cladribine, Crisnatol Mesylate, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin Hydrochloride, Decitabine,Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquone, Docetaxel,Doxorubicin, Doxorubicin Hydrochloride, Droloxifene, DroloxifeneCitrate, Dromostanolone Propionate, Duazomycin, Edatrexate, EflomithineHydrochloride, Elsamitrucin, Enloplatin, Enpromate, Epipropidine,Epirubicin Hydrochloride, Erbulozole, Esorubicin Hydrochloride,Estramustine, Estramustine Phosphate Sodium, Etanidazole, Ethiodized OilI 131, Etoposide, Etoposide Phosphate, Etoprine, FadrozoleHydrochloride, Fazarabine, Fenretinide, Floxuridine, FludarabinePhosphate, Fluorouracil, Flurocitabine, Fosquidone, Fostriecin Sodium,Gemcitabine, Gemcitabine Hydrochloride, Gold Au 198, Hydroxyurea,Idarubicin Hydrochloride, Ifosfamide, Ilmofosine, Interferon Alfa-2a,Interferon Alfa-2b, Interferon Alfa-n1, Interferon Alfa-n3, InterferonBeta-Ia, Interferon Gamma-Ib, Iproplatin, Irinotecan Hydrochloride,Lanreotide Acetate, Letrozole, Leuprolide Acetate, LiarozoleHydrochloride, Lometrexol Sodium, Lomustine, Losoxantrone Hydrochloride,Masoprocol, Maytansine, Mechlorethamine Hydrochloride, MegestrolAcetate, Melengestrol Acetate, Melphalan, Menogaril, Mercaptopurine,Methotrexate, Methotrexate Sodium, Metoprine, Meturedepa, Mitindomide,Mitocarcin, Mitocromin, Mitogillin, Mitomalcin, Mitomycin, Mitosper,Mitotane, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole,Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Peliomycin,Pentamustine, Peplomycin Sulfate, Perfosfamide, Pipobroman, Piposulfan,Piroxantrone Hydrochloride, Plicamycin, Plomestane, Porfimer Sodium,Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin,Puromycin Hydrochloride, Pyrazofurin, Riboprine, Rogletimide, Safingol,Safingol Hydrochloride, Semustine, Simtrazene, Sparfosate Sodium,Sparsomycin, Spirogermanium Hydrochloride, Spiromustine, Spiroplatin,Streptonigrin, Streptozocin, Strontium Chloride Sr 89, Sulofenur,Talisomycin, Taxane, Taxoid, Tecogalan Sodium, Tegafur, TeloxantroneHydrochloride, Temoporfin, Teniposide, Teroxirone, Testolactone,Thiamiprine, Thioguanine, Thiotepa, Tiazofurin, Tirapazamine, TopotecanHydrochloride, Toremifene Citrate, Trestolone Acetate, TriciribinePhosphate, Trimetrexate, Trimetrexate Glucuronate, Triptorelin,Tubulozole Hydrochloride, Uracil Mustard, Uredepa, Vapreotide,Verteporfin, Vinblastine Sulfate, Vincristine Sulfate, Vindesine,Vindesine Sulfate, Vinepidine Sulfate, Vinglycinate Sulfate,Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate,Vinzolidine Sulfate, Vorozole, Zeniplatin, Zinostatin, and ZorubicinHydrochloride.

The herein provide composition can further comprise one or moreradiosensitizers, including, but not limited to, gemcitabine,5-fluorouracil, pentoxifylline, and vinorelbine.

5. Carriers

The disclosed mammalian ECM can be combined, conjugated or coupled withor to carriers and other compositions to aid administration, delivery orother aspects of the ECM and their use. For convenience, suchcompositions are referred to herein as carriers. Carriers can, forexample, be a small molecule, pharmaceutical drug, fatty acid,detectable marker, conjugating tag, nanoparticle, or enzyme.

The disclosed compositions can be used therapeutically in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the composition, without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe composition, which matrices are in the form of shaped articles,e.g., films, liposomes or microparticles. It will be apparent to thosepersons skilled in the art that certain carriers may be more preferabledepending upon, for example, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders can be desirable.

Some of the compositions can potentially be administered as apharmaceutically acceptable acid- or base- addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

D. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted. For example, U.S. Pat. No. 5,275,826, U.S.Pat. No. 5,554,389, U.S. Pat. No. 6,099,567, and U.S. Pat. No.6,379,710, are disclosed herein by reference for methods of makingcompositions comprising small intestine submucosa (SIS), urinary bladdersubmucosa (UBS), stomach submucosa (SS), and liver submucosa (LS) orliver basement membrane (LBM), respectively.

E. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in .degree. C. oris at ambient temperature, and pressure is at or near atmospheric.

1. Example 1 Retrospective Evaluation of New Onset Postoperative AtrialFibrillation in Patients Receiving the CorMatrix® ECM®

A retrospective, multi-center, two-arm, chart review was conducted inwhich the CorMatrix® ECM™ was utilized. The objective of thisretrospective trial was to assess whether utilization of the CorMatrix®ECM® to reconstruct the normal pericardial barrier can result in a lowerrate of new onset postoperative atrial fibrillation as compared topatients who did not undergo pericardial closure.

CorMatrix® ECM® can be used for the reconstruction and repair of thepericardium following open heart surgery. Intact, the pericardiumprovides passive restraint to the heart preventing over dilation andhelping to modulate abrupt volumetric changes. By reconstructing thepericardium with the CorMatrix® ECM®, the natural pericardial restraintcan be restored. The purpose of this retrospective clinical trial was toassess if there is a reduction observed in new onset postoperativeatrial fibrillation by analyzing patients who had their nativepericardium reconstructed with the CorMatrix® ECM® as compared to thosewho did not undergo pericardial closure following isolated coronaryartery bypass graft (CABG) procedures.

The CorMatrix® ECM® was supplied in four-ply sheets of variousdimensions, which can be cut to size as the physician deems necessaryfor the procedure.

The definition of new onset postoperative atrial fibrillation used forthis retrospective study is based on the definition used in the Societyof Thoracic Surgeons (STS) Adult Cardiac Surgery Database 2007. Thedefinition is as follows: “Indicate whether the patient had a new onsetof Atrial Fibrillation/Flutter (AF) requiring treatment. Does notinclude recurrence of AF which had been present preoperatively. Do notinclude patients that had preoperative atrial fibrillation (treated ornon-treated). The event must be of new origin.

All patients were required to meet the following inclusion criteria inorder to be included as part of this retrospective clinical trial: thiscardiac operation was the subject's first or primary cardiac operation,and the subject must have undergone an isolated CABG procedure.

Patients were not included as part of this retrospective clinical trialif one or more of the following exclusion criteria is met: prior historyof atrial fibrillation, prior history of open heart surgery, priorhistory of pericarditis, prior history of amiodarone in the past sixmonths, and concomitant valve surgery planned.

Patients who had their native pericardium reconstructed with theCorMatrix® ECM® had a statistically significant decrease in theincidence of A-fib as compared to those who did not undergo pericardialclosure following isolated CABG procedures. The usual incidence of A-fibis around 25%. For these studies, the A-fib incidence was between 4% and8% (1/25 and 4/52).

2. Example 2 Modulation of Cardiac Remodeling with Acellular MatrixEmulsion is Associated with Myofibroblast Proliferation and Angiogenesisvia Recruiting C-kit Positive Cells after Myocardial Infarction

Degradation of native extracellular matrix (ECM) has been associatedwith maladaptive cardiac remodeling after infarction. As shown herein,xenogeneic acellular matrix emulsion injected into infarcted myocardiumpromoted myofibroblast proliferation and angiogenesis by recruiting hostc-kit positive cells.

Sixty-four rats were subjected to 45 minutes regional ischemia followedby 3, 7, 21 and 42 days of reperfusion. Histological examination wasperformed by immunohistological staining, and cardiac function wasanalyzed using echocardiography. ECM emulsion (30-50 .mu.l) was injectedinto the area at risk myocardium after reperfusion and localization ofthe emulsion was confirmed with Masson Trichome staining. At 7 days ofreperfusion, the population of c-kit positive cells within the emulsionarea increased significantly relative to the control (32.+−.0.6* vs.15.+−.3/1000 nuclei), consistent with significantly enhanced expressionof 31 kDa stem cell factor detected by Western blotting. Along with thischange, myofibroblasts accumulated in the emulsion region to asignificant extent compared to the control (59.+−.8* vs. 30.+−.3/HPF).Strong immunoreactivity of VEGF was observed in the emulsion area andangiogenesis was significantly enhanced relative to the control,evidenced by increased density of .alpha.-smooth muscle actin-positivevessels (70.+−.10* vs. 20.+−.4/HPF) and vWF-positive vessels (95.+−.14*vs. 34.+−.8/HPF), respectively. At 42 days of reperfusion,echocardiography showed improvements in end-systolic volume (0.3.+−.0.1*vs. 0.6.+−.0.3 ml)), fractional shortening (33.+−.5* vs. 24.+−.6%) andejection fraction (67.+−.6* vs. 53.+−.10%) in the emulsion group. Thewall thickness of the infarcted middle anterior septum in the emulsiongroup was also significantly greater than that in the Control(0.19.+−.0.02* vs. 0.15.+−.0.02 cm).

Intramyocardial injection of an acellular extracellular matrix emulsioninto the ischemic/reperfused myocardium attenuated maladaptive cardiacremodeling and preserved cardiac function, potentially mediated byenhanced myofibroblast proliferation and angiogenesis via recruitingc-kit positive cells. * p<0.05 emulsion vs. control.

What is claimed is:
 1. A method of treating atrial fibrillation in asubject, comprising: providing an extracellular matrix (ECM) compositioncomprising ECM from a mammalian tissue source comprising acellularbasement membrane, said ECM composition further comprising a statin andan exogenously added growth factor comprising transforming growthfactor-β (TGF-β), said statin and growth factor being linked to saidECM, wherein, when said ECM composition is administered to damagedmyocardium tissue of said subject, said statin and growth factorinteract with said ECM to modulate inflammation of said myocardiumtissue and induce remodeling of cardiac tissue, whereby said ECMcomposition modulates electrical activity in the heart of said subject;and delivering said ECM composition directly to said damaged myocardiumtissue of said subject, wherein said ECM composition reduces incidenceof atrial fibrillation.
 2. The method of claim 1, wherein said mammaliantissue source comprises a tissue source selected from the groupconsisting of mammalian tunica propria, tunica submucosa, tunicamuscularis and tunica serosa.
 3. The method of claim 1, wherein saidstatin is selected from the group consisting of lovastatin, simvastatin,atorvastatin, fluvastatin, pravastatin, rosuvastatin, cerivastatin andpitavastatin.
 4. The method of claim 1, wherein said ECM compositionfurther comprises an embryonic stem cell.